Biochemical impact of a disease-causing Ile67Asn substitution on BOLA3 protein

Iron-sulfur (Fe-S) cluster biosynthesis involves the action of a variety of functionally distinct proteins, most of which are evolutionarily conserved. Mutations in these Fe-S scaffold and trafficking proteins can cause diseases such as multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia, and mitochondrial encephalopathy. Herein, we investigate the effect of Ile67Asn substitution in the BOLA3 protein that results in the MMDS2 phenotype. Although the exact functional role of BOLA3 in Fe-S cluster biosynthesis is not known, the [2Fe-2S]-bridged complex of BOLA3 with GLRX5, another Fe-S protein, has been proposed as a viable intermediary cluster carrier to downstream targets. Our investigations reveal that the Ile67Asn substitution impairs the ability of BOLA3 to bind its physiological partner GLRX5, resulting in a failure to form the [2Fe-2S]-bridged complex. Although no drastic structural change in BOLA3 arises from the substitution, as evidenced by wild-type and mutant BOLA3 1H-15N HSQC and ion mobility native mass spectrometry experiments, this substitution appears to influence cluster reconstitution on downstream proteins leading to the disease phenotype. By contrast, substituted derivatives of the holo homodimeric form of BOLA3 are formed and remain active toward cluster exchange.